P53 protein is involved in regulation of the cell cycle, apoptosis and cell differentiation. Arrest at the G1/S checkpoint and progression through cell death are correlated with increases in p53 protein levels. Adoption of correct tertiary structure for DNA binding and transactivation functions are highly related to phosphorylation status of p53. While the rise and fall of total p53 levels are important to function, it does not reflect the dynamic changes in phosphorylation state. The purpose of this project is to study changes in p53 phosphorylation by separating different phosphorylated species by charge and mass separation using two dimensional gel electrophoresis (2D PAGE). Individual isoforms will be related to p53- mediated cellular functions in order to identify bioactive species of the protein. We have found 11 isoforms in human cells, which are all progressively phosphorylated except one isoform which appears to be the unmodified gene product. Studies using phosphorimage analysis permitted quantitation of individual human p53 isoforms and distinguished between high and low phosphorylated isoforms by normalizing against uniform incorporation of radiolabeled methionine. Examination of various cell types shows that >90% of p53 is phosphorylated. Since p53 is expressed at a low level in normal cells, we used a baculovirus mediated expression system in insect cells to isolate quantities sufficient for biochemical studies and antibody production. Comparison of recombinant p53 with wild type p53 from human epithelial cells showed the number of isoforms & 2D PAGE separation pattern to be the same. Development of the preceding analytical techniques and recombinant protein expression system will enable us to test the hypothesis that specific p53 isoforms are bioactive in wild type protein and non-functional in mutants. We are presently developing cell culture models for induction of p53-dependent responses for G1 arrest and apoptosis to produce and isolate bioactive p53 isoforms for molecular analyses.